Endoscopic septoplasty: Learning curve.

AIMS: The aim of the current study was to report the learning curve for endoscopic septoplasty for a senior surgeon already trained in endonasal sinus surgery. MATERIAL AND METHODS: From November 2011 to September 2012, 100 patients were prospectively included and grouped in 5 consecutive groups of 20 by date of surgery. The primary endpoint was operative time. Intra- and postoperative complications and functional assessment were also analyzed. RESULTS: Operative time decreased with the surgeon's experience and became stable after 60 procedures. Operative time saving was about 10min per 20 procedures. Mean operative time was stable between groups 4 (21.1±9.6min) and 5 (19.2±8.2min). There was a 2% rate of conversion to conventional surgery for technical problems. The number of procedures free of accidental mucosal lesion increased and became stable after 40 procedures. There was a 4% rate of residual postoperative perforation. Nasal Obstruction and Septoplasty Effectiveness (NOSE) score improved postoperatively in each group (P<0.05). CONCLUSION: After 60 endoscopic septoplasty procedures, a senior surgeon masters the surgical technique with satisfactory operative times, and a decreasing rate of intra- and postoperative complications.

Endoscopic vs. conventional septoplasty: A review of the literature.

The aim of this review of literature was to compare conventional and endoscopic septoplasty in terms of operating time, functional efficacy and perioperative morbidity. A systematic review of the scientific literature was performed on the PubMed database, Google and Google Scholar, searching for randomized prospective trials comparing endoscopic and conventional septoplasty. The primary endpoint was operating time, and the secondary endpoints were intra- and postoperative complications, postoperative pain, hospital stay and functional result. Twenty-nine articles published between 1991 and 2012 compared conventional and endoscopic septoplasty, five of which were prospective randomized trials. Operating time was shorter with endoscopic surgery (P<0.001), with less mucosal damage (P<0.01); there was less synechia (P<0.01) and residual deformity (P<0.05); and postoperative pain was milder. Endoscopic septoplasty thus shortened surgery time and reduced perioperative complications, but the functional result was the same as with conventional septoplasty.

Endoscopic septoplasty: Tips and pearls.

This article is designed to provide a step-by-step description of our endoscopic septoplasty technique and discuss its difficulties and technical tips. Endoscopic septoplasty comprises 10 steps: diagnostic endoscopy, subperichondral infiltration, left mucosal incision, dissection of the left subperichondral flap, cartilage incision (0.5 centimetre posterior to the mucosal incision), dissection of the right subperichondral flap, anterior cartilage resection, perpendicular plate dissection, dissection and resection of the maxillary crest, endoscopic revision, mucosal suture and Silastic stents. A satisfactory postoperative result was observed at 3 months in 97% of cases in this series. The main contraindication to endoscopic septoplasty is anterior columellar deviation of the nasal septum requiring a conventional procedure.

The TLR1/2 agonist PAM(3)CSK(4) instructs commitment of human hematopoietic stem cells to a myeloid cell fate.

Toll-like receptors (TLRs) constitute a family of nonpolymorphic receptors that are devoted to pathogen recognition. In this work, we have explored the impact of TLR ligands (TLR-L) on human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We show that HSCs and HPCs have a comparable pattern of expression of TLR transcripts characterized by the predominance of TLR1, -2, -3, -4 and -6. In long-term cultures of HSCs, HPCs and stromal cells, most TLR-L profoundly inhibited B-cell development while preserving or enhancing the production of myeloid cells. In short-term cultures, the TLR1/2 ligand PAM(3)CSK(4) induced a large proportion of HPCs to express markers of the myelomonocytic lineage. PAM(3)CSK(4) induced only marginal expression of myeloid lineage markers on HSCs but promoted their myeloid commitment as revealed by their acquisition of the phenotype of multi- and bipotential myeloid progenitors and by upregulation of the transcription factors PU.1, C/EBPalpha and GATA-1. Our results suggest that TLR agonists can bias the lineage commitment of human HSCs and shift the differentiation of lineage-committed progenitors to favor myelopoiesis at the expense of lymphoid B-cell development.

PUVA apoptotic response in activated and resting human lymphocytes.

In extracorporeal photopheresis (ECP) collected cells are treated by 8 methoxypsoralen and UVA (PUVA) which induced apoptosis. The mechanism of action of reinfused cell is unclear. A vaccination model postulates an efficient presentation of apoptotic alloreactive cells to the patient immune system. The efficiency may depend upon a predominance of apoptotic alloreactive cells after PUVA. Such selectivity could result from their activation. We studied apoptosis in resting and PHA-activated lymphocytes. Both were equally susceptible. Changes in early apoptosis were possibly missed. We evaluated the effect of preincubation before PUVA. During preincubation monocyte could affect lymphocytes susceptibility to apoptosis as an increase of number of apoptotic cells was observed after 72 hours in stimulated and resting cells. Our findings do not preclude a selectivity of other PUVA effects since expression of membrane marker also targets to PUVA is modified by activation.

Inhibition of thymidine synthesis by folate analogues induces a Fas-Fas ligand-independent deletion of superantigen-reactive peripheral T cells.

Methotrexate (MTX), a folate antagonist with multiple enzymatic targets, is used in the treatment of malignancies as well as in autoimmune and chronic inflammatory diseases, and ZD1694 (tomudex), a water-soluble quinazoline specific inhibitor of thymidylate synthase (TS), is used in the treatment of adenocarcinomas. In this study, we investigated the effects of these folate analogues on superantigen (SAg)-reactive peripheral T cells in vivo. In BALB/c mice, staphylococcal enterotoxin B (SEB)-induced cytokine secretion, IL-2R (CD25) expression and early deletion of a fraction of SEB-reactive V(beta)8(+) T cells were not impaired by either MTX (7 mg/kg/day) or tomudex (5 mg/kg/day). However, both MTX and tomudex prevented V(beta)8-selective T cell expansion and accelerated their peripheral elimination. Administration of thymidine (500 mg/kg/12 h) completely abrogated this effect, indicating that inhibition of TS but not that of other folate-dependent enzymes was the main mechanism involved. Furthermore, a marked increase of apoptotic cells restricted to the V(beta)8(+) T cell subset indicated that proliferation inhibition was associated with apoptosis. In contrast with peripheral V(beta)8(+) T cell deletion, MTX and tomudex did not prevent the increase of V(beta)8(+) thymocytes triggered by SEB. Experiments in C57BL/6-lpr/lpr mice further demonstrated that deletion of V(beta)8(+) T cells induced by folate analogues was independent of Fas-Fas ligand interaction. Our results provide evidence that folate analogues may selectively delete dividing peripheral T cells through TS inhibition, but do not interfere with other events triggered by SAg.

Role of acidic sphingomyelinase in Fas/CD95-mediated cell death.

Engagement of the Fas receptor has been reported to induce ceramide generation via activation of acidic sphingomyelinase (aSMase). However, the role of aSMase in Fas-mediated cell death is controversial. Using genetically engineered mice deficient in the aSMase gene (aSMase(-/-)), we found that thymocytes, concanavalin A-activated T cells, and lipopolysaccharide-activated B cells derived from both aSMase(-/-) and aSMase(+/+) mice were equally sensitive to Fas-mediated cell death, triggered by either anti-Fas antibody or Fas ligand in vitro. Similarly, activation-induced apoptosis of T lymphocytes was unaffected by the status of aSMase, and aSMase(-/-) mice failed to show immunological symptoms seen in animals with defects in Fas function. In vivo, intravenous injection of 3 microg/25 g mouse body weight of anti-Fas Jo2 antibody into aSMase(-/-) mice failed to affect hepatocyte apoptosis or mortality, whereas massive hepatocyte apoptosis and animal death occurred in wild type littermates. Animals heterozygous for aSMase deficiency were also significantly protected. Susceptibility of aSMase(-/-) mice to anti-Fas antibody was demonstrated with higher antibody doses (>/=4 microg/25 g mouse). These data indicate a role for aSMase in Fas-mediated cell death in some but not all tissues.

Protein kinase ctheta cooperates with calcineurin to induce Fas ligand expression during activation-induced T cell death.

Activation-induced cell death is mediated by the TCR-induced expression of the Fas ligand (FasL) on the surface of T cells, followed by binding to its receptor Fas. FasL expression is induced by stimulating T cells with a combination of phorbol ester and Ca2+ ionophore, implicating a role for protein kinase C (PKC) in this process. However, the precise mechanisms that regulate FasL expression, including the contribution of distinct T cell-expressed PKC isoforms, are poorly understood. Herein, we report that PKCtheta, a Ca2+-independent PKC isoform that we have previously isolated as a PKC enzyme selectively expressed in T cells, plays an important role in these processes. A constitutively active PKCtheta mutant preferentially induced FasL expression and activated the corresponding gene promoter; conversely, a dominant-negative PKCtheta mutant blocked FasL expression induced by anti-CD3 or PMA plus ionomycin stimulation. Furthermore, PKCtheta synergized with calcineurin to provide a potent stimulus for FasL promoter activation. Full activation of the promoter required its binding sites for the transcription factors NF-AT, AP-1, and NF-kappaB. The biological significance of these findings is implicated by the finding that rottlerin, a selective PKCtheta inhibitor, blocked FasL induction by anti-CD3 or PMA plus ionomycin stimulation and, consequently, protected human Jurkat T cells and the mouse T cell hybridoma A1.1 from activation-induced cell death.

Regulation of fas-ligand expression during activation-induced cell death in T lymphocytes via nuclear factor kappaB.

T cell receptor engagement activates transcription factors important for cytokine gene regulation. Additionally, this signaling pathway also leads to activation-induced apoptosis in T lymphocytes that is dependent on FasL transcription and expression. Here we demonstrate that nuclear factor kappaB (NF-kappaB), which is involved in the transcriptional regulation of many cytokine genes expressed in activated lymphocytes, also plays a role in T cell activation-induced FasL expression. Inhibition of NF-kappaB activity in a T cell hybridoma leads to decreased FasL expression and apoptosis upon T cell receptor stimulation. We identified the NF-kappaB site in the FasL promoter that contributes to such regulation. Co-expression of p65 (Rel A) with the FasL promoter enhanced its activity, and co-expression of IkappaB dramatically inhibited the inducible promoter activity. In contrast, the transcription factor AP-1 is not required for activation-induced FasL promoter activity. These results define a role for NF-kappaB in mediating FasL expression during T cell activation.

Transforming growth factor beta1 inhibits Fas ligand expression and subsequent activation-induced cell death in T cells via downregulation of c-Myc.

Activation-induced cell death (AICD) is a process that regulates the size and the duration of the primary immune T cell response. In this report, we investigated the mechanisms involved in the regulation of AICD by transforming growth factor beta1 (TGF-beta1). We found that TGF-beta1 decreased apoptosis of human T cells or T cell hybridomas after activation by anti-CD3. This decrease was associated with inhibition of Fas (Apo-1/CD95) ligand (FasL) expression, whereas Fas signaling was not affected by TGF-beta1. In parallel, TGF-beta1 inhibited c-Myc expression in T cell hybridomas, and ectopic expression of a chimeric molecule composed of c-Myc and the steroid binding domain of the estrogen receptor (Myc-ER) blocked both the inhibition of FasL and the decrease of AICD induced by TGF-beta1, providing that 4-hydroxytamoxifen was present. These results identify one mechanism by which TGF-beta1 blocks AICD to allow the clonal expansion of effector T cells and the generation of memory T cells during immune responses.

DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-kappa B and AP-1.

Apoptosis induced by DNA damage and other stresses can proceed via expression of Fas ligand (FasL) and ligation of its receptor, Fas (CD95). We report that activation of the two transcription factors NF-kappa B and AP-1 is crucially involved in FasL expression induced by etoposide, teniposide, and UV irradiation. A nondegradable mutant of I kappa B blocked both FasL expression and apoptosis induced by DNA damage but not Fas ligation. These stimuli also induced the stress-activated kinase pathway (SAPK/JNK), which was required for the maximal induction of apoptosis. A 1.2 kb FasL promoter responded to DNA damage, as well as coexpression with p65 Rel or Fos/Jun. Mutations in the relevant NF-kappa B and AP-1 binding sites eliminated these responses. Thus, activation of NF-kappa B and AP-1 contributes to stress-induced apoptosis via the expression of FasL.

Immunosuppressive properties of methotrexate: apoptosis and clonal deletion of activated peripheral T cells.

The folate antagonist methotrexate (MTX) is extensively used in graft-versus-host disease, rheumatoid arthritis, and other chronic inflammatory disorders. In addition to its antiinflammatory activity associated with increased release of adenosine, MTX exerts antiproliferative properties by inhibition of dihydrofolate reductase and other folate-dependent enzymes. However, the mechanisms of immunosuppressive properties associated with low-dose MTX treatments are still elusive. We report here that MTX (0.1-10 microM) induces apoptosis of in vitro activated T cells from human peripheral blood. PBL exposed to MTX for 8 h, then activated in drug-free medium, underwent apoptosis, which was completely abrogated by addition of folinic acid or thymidine. Apoptosis of activated T cells did not require interaction between CD95 (Fas, APO-1) and its ligand, and adenosine release accounted for only a small part of this MTX activity. Apoptosis required progression of activated T cells to the S phase of the cell cycle, as it was prevented by drugs or antibodies that interfere with IL-2 synthesis or signaling pathways. MTX achieved clonal deletion of activated T cells in mixed lymphocyte reactions. Finally, in vitro activation of PBL taken from rheumatoid arthritis patients after MTX injection resulted in apoptosis. Altogether, the data demonstrate that MTX can selectively delete activated peripheral blood T cells by a CD95-independent pathway. This property could be used as a new pharmacological end point to optimize dosage and timing of MTX administration. It may account for the immunosuppressive effects of low-dose MTX treatments.

Induction of Fas (Apo-1, CD95)-mediated apoptosis of activated lymphocytes by polyclonal antithymocyte globulins.

Polyclonal horse antilymphocyte and rabbit antithymocyte globulins (ATGs) are currently used in severe aplastic anemia and for the treatment of organ allograft acute rejection and graft-versus-host disease. ATG treatment induces a major depletion of peripheral blood lymphocytes, which contributes to its overall immunosuppressive effects. Several mechanisms that may account for lymphocyte lysis were investigated in vitro. At high concentrations (.1 to 1 mg/mL) ATGs activate the human classic complement pathway and induce lysis of both resting and phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells. At low, submitogenic, concentration ATGs induce antibody-dependent cell cytotoxicity of PHA-activated cells, but not resting cells. They also trigger surface Fas (Apo-1, CD95) expression in naive T cells and Fas-ligand gene and protein expression in both naive and primed T cells, resulting in Fas/Fas-L interaction-mediated cell death. ATG-induced apoptosis and Fas-L expression were not observed with an ATG preparation lacking CD2 and CD3 antibodies. Susceptibility to ATG-induced apoptosis was restricted to activated cells, dependent on IL-2, and prevented by Cyclosporin A, FK506, and rapamycin. The data suggest that low doses of ATGs could be clinically evaluated in treatments aiming at the selective deletion of in vivo activated T cells in order to avoid massive lymphocyte depletion and subsequent immunodeficiency.

Caspase-dependent ceramide production in Fas- and HLA class I-mediated peripheral T cell apoptosis.

We recently demonstrated that the engagement of HLA class I alpha1 domain induced Fas-independent apoptosis in human T and B lymphocytes. We analyzed the signaling pathway involved in HLA class I-mediated apoptosis in comparison with Fas (APO-1, CD95)-dependent apoptosis. The mouse mAb90 or the rat YTH862 monoclonal antibodies which bind the human HLA class I alpha1 domain induced the production of ceramide which was blocked by addition of the phosphatidylcholine-dependent phospholipase C inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved at least two different caspases, an interleukin-1 converting enzyme-like protease and another protease inhibited by the CPP32-like protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA class I signaling pathways, we failed to demonstrate any physical association between these two molecules. We also report that the pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane potential and generation of ceramide induced by anti-HLA class I and anti-Fas monoclonal antibodies, whereas all three peptides efficiently inhibited apoptosis. Altogether these results suggest that signaling through Fas and HLA class I involve caspase(s), targeted by zVAD-fmk, which act upstream of ceramide generation and mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the mitochondria.